July 31st 2014

First load the required packages and specify the input file path.
We are going to use a sequence from human as input, which has been included as
as fasta file in the CRISPRseek package.
To perform off target analysis, we need to load Human BSgenome package
To annotate the target and off-targets, we need to load Human Transcript package
Additionaly, need to specify the file containing all restriction enzyme (RE) cut
patterns. You have the option to use the RE pattern file in the CRISPR package,
or specify your own RE pattern file.
Furthermore, you need to specify the output directory which will be the
directory to look for all the output files.

Scenario 5: Target and off-target analysis for user specified gRNAs

Calling the function offTargetAnalysis with findgRNAs = FALSE results in target
and off-target searching, scoring and annotating for the input gRNAs. The gRNAs
will be annotated with restriction enzyme cut sites for users to review later.
However, paired information will not be available.

without running time-consuming off-target analysis on all possible gRNAs.

Below is an example to search for all gRNAs that target at least one of the
alleles. Two files are provided containing sequences that differ by a single
nucleotide polymorphism (SNP). The results are saved in file
scoresFor2InputSequences.xls in outputDir directory.

Excercise 3

Constraint gRNA Sequence by setting gRNA.pattern to require or exclude specific
features within the target site.

3a. Synthesis of gRNAs in vivo from host U6 promoters is more efficient if the
first base is guanine. To maximize the efficiency, what can we set gRNA.pattern?

3b. Synthesis of gRNAs in vitro using T7 promoters is most efficient when the
first two bases are GG. To maximize the efficiency, what can we set gRNA.pattern?

3c. Five consecutive uracils in any position of a gRNA will affect transcription
elongation by RNA polymerase III. To avoid premature termination during gRNA
synthesis using U6 promoter, what can we set gRNA.pattern?

3d. Some studies have identified sequence features that broadly correlate with
lower nuclease cleavage activity, such as uracil in the last 4 positions of
the guide sequence. To avoid uracil in these positions, what can we specify
gRNA.pattern?

Excercise 4

In the examples we went through, we deliberately restricted searching off-targets
in chromosome X. If we are interested in genome-wide search, what should we set
chromToSearch to?

Excercise 5

Find gRNAs in a paired configration with distance apart between 5 and 15 without
performing off-target analysis

Excercise 6

Create a transcriptDB object

Excercise 7

It is known that different CRISPR-cas system uses different PAM sequence, what
parameter needs to be reset?

Excercise 8

It is known that different CRISPR-cas system has different gRNA length, what
parameter needs to be reset?

Excercise 9

Which parameter needs to be reset to 8 if we are interested in finding gRANs with
restriction enzyme pattern of size 8 or above?

Excercise 10

New penalty matrix has been recently derived, which parameter needs to be set
accordingly?

Excercise 11

It has been shown that although PAM sequence NGG is preferred, a variant NAG is
also recognized with less effecieny. The researcher is interested in performing
off-target searching to include both NGG and NAG variants, but requiring that
gRNAs must precede NGG. What parameter(s) need to be set correctly to carry such
a search?